During hematopoiesis, the cell cycle and differentiation must be tightly regulated. Defects in regulation can lead to anemia or leukemia. Protein degradation by the ubiquitin pathway regulates critical regulators of hematopoiesis (e.g. p27, p21, p130, AML1/Runx1, and the Jak2 kinase). The Cul-4A cullin is part of an E3 ubiquitin ligase. Previous studies show that deletion of CUL-4A is embryonic lethal in vivo and its overexpression in granulocyte/macrophage-committed precursor cells and erythroblasts interferes with cell cycle exit and terminal differentiation. Also, CUL-4A haploinsufficiency in affects normal hematopoiesis in vivo. Therefore, we hypothesize that heterozygous or null CUL-4A embryonic stem (ES) cells have abnormal hematopoiesis. Whether CUL-4A +/- and/or CUL-4A -/- ES cell lines exhibit a defect during in vitro differentiation into hematopoietic cells will be tested (Aims 1 and 2). RT-PCR will be used to measure the expression of hematopoietic lineage-restricted genes. For each lineage whose differentiation is found to be abnormal, the differentiation stage where CUL-4A is required will be determined (Aim 3). Also, CUL-4A expression when wild type ES cells are induced to differentiate along this/these lineage(s) will be measured.